This invention relates to recombinant microorganisms (i.e., bacteria, yeast, and fungi) that display an oligosaccharide-comprising binding moiety that can compete with a ligand for binding to a receptor for the ligand, and use of the microorganisms to deliver the oligosaccharide to a human or other animal. The microoganism can be used for adsorbing toxins or pathogenic microorganisms from a particular environment.
Surfaces of all cells express a complexity of oligosaccharides that provide a number of functions. A primary one of these functions is determining on their own or together with other molecules, interactions with other cells or molecules. The nature, linkage and conformation of sugar residues of the oligosaccharides, and in particular residues at or close to the non-reducing terminus of an oligosaccharide, determines whether the oligosaccharide will or will not participate in a particular receptor-ligand interaction.
The susceptibility of an animal to infection and the eventual physiological site affected by an infection is to a large extent determined by the expression on the cell surface of such oligosaccharide receptors. In the case of enteric infections, one primary prerequisite for pathogenesis is that the microorganism persists in the intestinal lumen of the host. Generally this requires some form of adherence to the lumeinal epithelium (forming the mucosal surface of the gut), otherwise the micro-organism is flushed from the gut. Additionally for a toxigenic organism, the toxin also needs to bind to the luminal epithelium and, for some toxins, needs to be absorbed systemically to be effective, otherwise, it too would be flushed from the gut.
Certain surface structures of pathogenic and other bacteria known as adhesins mediate adherence to luminal epithelial cells. A number of adhesins are known and organisms without adhesins are generally of low virulence. Adhesins are proteinaceous factors which promote the adherence of bacteria and viruses to cells of their hosts. Adhesins can be either fimbrial or filamentous in structure or they may be afimbrial. Adhesins associated with fimbriae may be associated with accessory proteins such as tip proteins at an extreme end of the fimbrial structure and such tip proteins can be regarded as lectins. The receptor on the host cell has in some cases been determined and shown to be a carbohydrate such as an oligosaccharide associated with a glycolipid or a glycoprotein.
The best characterised system from a molecular and biological viewpoint, is the P-fimbriae (also called pili) produced by uropathogenic Escherichia coli. This tip adhesin binds the glycolipid Gb3 (globotriaosyl ceramidexe2x80x94see below) and the fimbrial subunits can be purified by affinity chromatography using a Gb3 mimic. Another well characterised group of adhesins are those associated with enterotoxigenic E. coli (ETEC) strains which infect pigs to cause scours. These are termed the K88 type and a number of variants are known, being K88ab, K88ac and K88ad. The adhesins associated with these fimbriae have been shown to have different receptor requirements, which includes the presence of both glycolipid and protein receptors. The carbohydrate requirement has been characterised in at least some of these, for example K88ad uses carbohydrates of the lactoneotetraose series of glycolipids.
A number of toxins have been identified and include, Shiga toxins (also referred to as Shiga-like toxins and verotoxins), toxins produced by various species of Clostridia, including tetanus toxin, botulinum toxin, and C. diffcile toxins A and B, Staphylococcal enterotoxins, Escherichia coil heat labile and heat stable enterotoxins and cholera toxin. Without toxin activity the majority of otherwise enterotoxigenic bacteria would be less capable of causing disease.
The receptors for the majority of adhesins and toxins identified to date are carbohydrate in nature. For example, the glycolipid globotriaosyl ceramide (Gb3) which has the structure Galxcex1[1xe2x86x924]Galxcex2[1xe2x86x924]Glc-ceramide, is the preferred receptor for most members of the Shiga toxin family. Similarly, the ganglioside GM1 is the receptor for cholera toxin and E. coli heat labile enterotoxin type I. C. difficile toxin A binds to several host receptors, all of which have in common a Galxcex2[1xe2x86x924]GlcNAc moiety. The neurotoxin produced by C. botulinum is also believed to be specific for a sialic acid containing glycoprotein or glycolipid present on neurons. The terminal Galxcex1[1xe2x86x924]Galxcex2 moiety present on Gb3 is the receptor for P pili, the major adhesin of uropathogenic E. coli strains. Similarly, asialo-GM1 is the receptor for adhesive pili (CFAs) of some enterotoxigenic E. coli strains. The sialated gangliosides NeuGc-GM3 and NeuNAc-GM3 have also been identified as the target cell receptors for porcine rotavirus strains, and it is presumed that rotavirus strains causing disease in humans also bind specific oligosaccharide moieties present on cell surface glycolipids.
The elucidation of the nature of oligosaccharides acting as receptors for particular toxins and pathogenic microorganisms has opened up a promising avenue in the diagnosis and potential treatment or prevention of diseases caused by these agents. The use of the sugar residues forming receptors for toxins or adhesins has been proposed as a means of specifically identifying the toxins or bacteria involved in an infection. For example, the ganglioside receptor GM1 is used as a specific capturing agent in ELISA assays for the presence of cholera toxin.
It has also been proposed to use synthetically prepared oligosaccharides as a means of adsorbing toxins or the like from samples. Examples of proposed uses of receptors for adsorbing toxins or pathogenic organisms out of a sample include Krivan et al in U.S. Pat. No. 5,696,000 which discusses the pharmaceutical use of certain tetra- and tri- saccharide receptors coupled to a carrier such as a liposome to inhibit the adherence of micro-organisms to susceptible cells. A similar use for toxins such as Shiga toxin, can be seen in U.S. Pat. No. 5,849,714 to Rafter et al which discloses the use of a synthetic construct of sugar residues making up the globotriose receptor, coupled by a linker to an inert support for use in treatment of bacterial dysentery.
A problem arises, however, in the synthesis and delivery of these compounds, because oligosaccharides are difficult or expensive to synthesise chemically, the conformation may not be appropriate, and the oligosaccharide may preferably need to be presented in an immobilized (non-diffusible) form. Thus, there is a need to provide a mechanism for delivery and presentation of the oligosaccharide moiety in an appropriate conformation in the environment where the toxin or pathogenic organism is to be adsorbed (for example the gastrointestinal tract).
The invention provides, in a first embodiment, a recombinant microorganism that displays on its surface a binding moiety that, when administered to an animal, competes with a ligand for binding to a receptor for the ligand. The binding moiety includes an oligosaccharide that is composed of at least one sugar residue that is attached to an acceptor moiety by a glycosyltransferase that is encoded by an exogenous nucleic acid which is present in the microorganism. The oligosaccharide can further include at least a second sugar residue that is attached to the acceptor moiety by at least a second glycosyltransferase. One or more of the additional glycosyltransferases can also be encoded by one or more exogenous nucleic acids that are present in the microorganism.
The receptor is typically present on a surface of a cell. Cells of interest include, for example epithelial or endothelial cells, in particular those that are present in an animal mucosal membrane.
The binding moiety is, in some embodiments, a mimic of a receptor for a toxin or adhesin of a pathogenic organism. Examples of toxins include, but are not limited to, enterotoxins, including shiga toxins, clostridial toxins, cholera toxins, E. coli enterotoxins, and Staphylococcal enterotoxins. In other embodiments, the binding moiety is a mimic of an adhesin receptor. Adhesins of interest include, but are not limited to, a CFA adhesin of an enterotoxigenic E coli., E. coli CS3 pili, K88ad fimbriae, an adhesin of Entamoeba histolyticum, and an adhesin of a virus.
In some embodiments, the binding moiety competes with a pathogenic organism for binding to a corresponding receptor on an animal epithelial or endothelial cell. Pathogenic organisms of interest include, for example, Staphylococcus pneumonia, H. influenza, H. parainfluenza, Chlamydia trachomatis, Acanthamoeba, Candida albicans, Helicobacter pylori and Pseudomonas spp.
In other embodiments, the binding moiety is a mimic of a receptor for a cell or molecule involved in inflammation. For example, the binding moiety can include a 3xe2x80x2-sialoside or a 6xe2x80x2-sialoside. Sialyl Lewisx and sialyl Lewisa are other examples of oligosaccharide structures that can function as a receptor mimic. Microorganisms that display these molecules can compete with leukocytes, for example, for binding to endothelial cells in the vasculature or other tissues and thus inhibit inflammation.
The invention also provides a recombinant microorganism expressing one or more exogenous sugar transferases, or one or more exogenous nucleotide sugar precursor synthesizing enzymes, said microorganism also expressing an acceptor molecule, said one or more exogenous sugar transferases being specific for the transfer of one or more sugar residues represented progressively from a non reducing terminal end of a receptor of either a toxin or an adhesin of a pathogenic organism, the exogenous sugar transferases progressively transferring said one or more sugar resides onto the acceptor molecule to thereby form a chimeric carbohydrate molecule with an exposed receptor mimic, said sugar precursor enzymes forming nucleotide precursors that are transferred to said acceptor molecule to make up said chimeric carbohydrate, said exposed receptor mimic capable of binding the toxin or the adhesin.
Also provided by the invention are preparations for administration to a mucosal surface. The preparations include a delivery microorganism or a partially or fully purified non-toxic preparation of a carbohydrate molecule therefrom. At least a part of the carbohydrate molecule generally acts as an exposed receptor mimic. For example, the receptor mimic can be capable of binding a toxin or an adhesin of a pathogen that normally binds to the mucosal surface. The receptor mimic is generally carried in a pharmaceutically acceptable excipient.
The invention also provides methods of administering a receptor mimic to a mucosal surface of a mammal. The methods involve the administration of a quantity of a delivery microorganism, or parts thereof, the delivery microorganism exhibiting one or more sugars in a configuration to form an exposed receptor mimic, the receptor mimic being a mimic of a receptor of a pathogen, said quantity being sufficient to reduce adherence of the pathogen or a toxin produced by the pathogen to the mucosal surface.
In another embodiment, the invention provides methods of testing for the presence of a toxin or a pathogenic microorganism in a sample. These methods involve, for example, contacting a sample with the purified carbohydrate as described above, with either the purified carbohydrate or the sample being immobilized. Unbound purified carbohydrate or toxin or pathogenic microorganism is washed off, and a detection means is added to detect bound purified carbohydrate and the toxin or pathogenic microorganism.